Mapping of DnaA, HU, FIS, IHF and H-NS on the origin of E. coli chromosome in vitro and in vivo

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The initiation of chromosomal DNA replication is at the basis of the duplication of every living cell. This process is highly regulated in both prokaryotic and eukaryotic cells and depends on the nutrients available, on cell to cell contacts, etc. In the bacteria Escherichia coli this process has been extensively studied in recent years. In the origin of replication (oriC) a sequence of about 350 bp binds several bacterial proteins including DnaA and several small chromosomal proteins like HU, IHF, FIS and H-NS. It is not yet understood how these proteins assemble together or how the initiation step is controlled.

The French and the German participating groups have been studying the association of these proteins with the origin sequence in vitro. On the other hand the Russian group has long-standing experience of inducing chemical cross-linking of proteins to DNA in vivo and in mapping the bending sites of these proteins. In a joint effort the purpose will be to study by such chemical cross-linking the nature of these proteins and their sites of bending on the origin sequence in vivo.

The availability of E.coli mutants lacking one or the other of these proteins as well as plasmids carrying the origin of replication of E. coli (oriC) wild type or mutated in different sites will be very helpful in correlating the structure of a native oriC complex containing the specific proteins positioned on their specific sites with its function. Furthermore, the possibility of following the assembly of the complex in vivo will certainly be helpful in understanding the control of the initiation in vivo.